Oligonucleotides are useful as diagnostic probes for the detection of "target" DNA or RNA sequences. A probe generally contains a sequence of ribo- or deoxyribonucleic acid complementary to the target sequence and a means for detection. A probe in which the nucleotides of the sequence contain natural .beta.-arabinose rather than ribose, together with a procedure which utilizes an anti-arabinose antibody as the means of detection, is proposed by McCormick, R. M., U.S. Pat. No. 4,760,017, filed Dec. 23, 1985, issued Jul. 26, 1988.
Oligonucleotides may also be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus or a bacterium. In nature, some RNA expression in bacteria is controlled by "antisense" RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity. A variety of recent studies using plasmid vectors for the introduction of antisense RNAs into eukaryotic cells have shown that they effectively inhibit expression of mRNA targets in vivo; see Green, P. J., et al. Ann. Rev. Biochem. 55:569-597 (1986). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment, which binds to the mRNA and prevents its translation into protein on ribosomes. See Paterson, B. M., et al., Proc. Natl. Acad. Sci. 74:4370-4374 (1977); and Hastie, N. D., et al., Proc. Natl. Acad. Sci. 75:1217-1221 (1978).
It has been shown that an appropriate small antisense oligonucleotide probe can inhibit replication of Rous Sarcoma Virus (RSV) in cell culture, as reported by Zamecnik, P. C., et al., Proc. Natl. Acad. Sci. USA 75:280 (1978), and that RSV viral RNA translation is inhibited under these conditions, as reported by Stephenson, et al., Proc. Natl. Acad. Sci. USA 75:285-288 (1978). It has also been shown that oligonucleotides complementary to a mRNA splice acceptor site in the HIV virus genome (the causative agent of AIDS) are capable of inhibiting expression and replication in cell culture; see Zamecnik, P. C., et al., Proc. Natl. Acad. Sci. USA 83:4143 (1986); Goodchild, et al., Proc. Natl. Acad. Sci USA 85:5507-5511 (1988).
Uncharged methylphosphonate oligodeoxynucleotides with a sequence complementary to the initiation codon regions of rabbit globin mRNA inhibited the translation of the mRNA in both cell-free systems and in rabbit reticulocytes, as reported by Blake, K. R., et al., Biochemistry 24:6139-6145 (1985). Another uncharged methylphosphonate oligonucleotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of Herpes simplex virus, Type 1, can inhibit virus replication in intact Vero cells. However, fairly high concentrations (&gt;25 .mu.M) of this nonionic probe were required for this inhibition.
In studies of the properties of oligomers composed of the .alpha.-deoxy-anomers of the natural .beta.-deoxynucleosides, it has been shown that oligo-(.alpha.-thymidylate) binds to polyadenylate much more strongly than the natural .beta.-oligomer, as reported by Thuong, N. T., et al., Proc. Natl. Acad. Sci. USA 84:5129-5133 (1987). This .alpha.-deoxyoligomer is also highly nuclease resistant, as reported by Cazenave, C., et al., Nucleic Acids Res. 15:10507-10521 (1987). Another .alpha.-oligomer, .alpha.-deoxy-(G.sub.2 T.sub.12 G.sub.2) has been shown to bind to (dA).sub.12 with a T.sub.m of 53.degree., whereas the corresponding oligo-(.beta.-anomer) binds with a T.sub.m of 27.degree.; see Gagnor, C., et al., Nucleic Acids Res. 15:10419-10436 (1987). Additionally, the antisense .alpha.-oligodeoxynucleotide binds parallel to the sense strand; see Sun, J., et al., Nucleic Acids Res. 15:6149-6158 (1987); and Morvan, F., et al., Nucleic Acids Res. 15:7027-7044 (1987). However, while .alpha.-deoxyribofuranosides provide nuclease resistance and have a greater hybrid stability as compared to the corresponding .beta.-oligomers, they are very difficult to synthesize because the .alpha.-anomers of the deoxynucleosides cannot be isolated in nature and are difficult and expensive to prepare synthetically
Therefore, the difficulty remained to find oligonucleotides which were easy to synthesize, were nuclease resistant, exhibited hybrid stability, and had improved hybridization efficiency.